Mosquitoes are the insect vectors for numerous blood-borne human diseases that cause millions of deaths each year worldwide. Mosquito larvae are filter-feeders that inhabit quiet open-water environments including marshes, ponds, and eddies along streams and rivers. Depending upon species, the larvae live at various depths in the water column and may be controlled by adding insecticides to the water. Some forms of bacterial toxins from Bacillus thuringiensis subspecies israelensis (Bti toxin) have strong dipteran activity, and are specific for mosquito and related fly larvae. Bti-toxins are considered safer than all chemical insecticides, because they are harmless to most other insect species and to humans and other mammals. BtBooster (BtB) proteins have been identified that enhance the toxicity of Bt-related toxins, and act by increasing toxin uptake in the larval midgut. A novel and inexpensive system for Bti and BtB production and delivery is needed that will reach diverse mosquito species in various aquatic environments and at various depths. Our biotechnical hypothesis is that: Bti and BtB expressed in seed oil bodies of oil-rich crop plants can be delivered effectively as a milled Bt- flour that will float at various levels in the water column and kill feeding mosquito larvae. In this Phase I application, we will test this hypothesis by expressing Bti and BtB in the oil-rich seeds of the model plant, Arabidopsis thaliana, a close relative of canola. The seeds will be milled into flour with a particle size approximating that of the food consumed by filter-feeding mosquito larvae. The buoyant Bti- and BtB-rich flour will be dispersed on water and fed to larvae of the various mosquito species that act as vectors for malaria, West Nile virus, and yellow fever and their mortality quantified. This project develops a more efficient, inexpensive, and environmentally safe solution to production and delivery of Bti-related toxins and Bt-Boosters for mosquito control. A phase II application is planned in which Bti and BtB will be expressed in canola (Brassica napus). The Bti/BtB expressing canola seeds can be produced and milled into Bt-flour at relatively low cost. PUBLIC HEALTH RELEVANCE Mosquitoes are vectors for numerous blood-born diseases, including malaria, yellow fever, West Nile virus, filariasis, Japanese encephalitis, and dengue fever and worldwide cause millions of deaths annually. This project is designed to develop a novel, more efficient, inexpensive, and environmentally safe solution to production and delivery of Bti-related toxins and Bt-Boosters for mosquito control at the larval stage, reaching diverse mosquito species in various aquatic environments. It should produce significant public health-related benefits through better control of vector-borne disease and substantial commercial opportunities. [unreadable] [unreadable] [unreadable]